Imaging Cytoskeleton Components by Electron Microscopy

Svitkina, Tatyana

doi:10.1007/978-1-0716-1661-1_2

Cited by 20 publications

(13 citation statements)

References 92 publications

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“…Further studies are needed to address various cytoskeletal proteins, especially investigating the role of protein 4.1B (37, 58, 59), which was not included in the study due to a lack of a commercial antibody apt for super resolution microscopy. A combination of electron microscopy with super-resolution microscopy techniques (24, 64, 65) might confirm and further investigate the underlying histopathological changes. Furthermore, the effect of cytoskeletal damage on nodal ion channels needs to be further investigated on a nanoscale level in future studies.…”

Section: Discussionmentioning

confidence: 99%

Super-resolution imaging pinpoints ultrastructural changes at the node of Ranvier in patients with polyneuropathy

Appeltshauser

Linke

Heil

et al. 2022

Preprint

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The peripheral node of Ranvier is the key element in saltatory conduction along myelinated axons, but its ultrastructural protein arrangement and interactions remain elusive in humans. To assess both physiological protein arrangement and pathological ultrastructural changes at the paranodal region of the node of Ranvier, we implement the super resolution microscopy method direct Stochastic Optical Reconstruction Microscopy (dSTORM) assisted by high-content confocal microscopy on nerve biopsies of patients with polyneuropathies. We demonstrate that a ~190nm periodic arrangement of axonal cytoskeletal proteins is conserved in humans, but impaired at the paranode in polyneuropathy. In patients with polyneuropathy, the periodic protein distances of the paranodal adhesion molecules Caspr-1 and neurofascin-155 were severely elongated, implying a lateral decoiling and partial loss of the axoglial complex at the node of Ranvier. Dual-color colocalization analysis of the super-resolved dSTORM images revealed that the axoglial complex itself remained closely attached even under pathological conditions, but was detached from its cytoskeletal axonal anchor. Correspondingly, high-content confocal analysis of nodes of Ranvier showed that paranodal elongation occurred especially in acute and severe axonal neuropathy. Our findings show that super-resolution techniques open the possibility to identify underlying nanoscale pathology in polyneuropathies, with possible future use in diagnostic assessment.

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Section: Discussionmentioning

confidence: 99%

Super-resolution imaging pinpoints ultrastructural changes at the node of Ranvier in patients with polyneuropathy

Appeltshauser

Linke

Heil

et al. 2022

Preprint

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“…To use a natural, rather than in vitro , system, we turned to cell cultures subjected to detergent extraction during fixation. This procedure results in the preservation of actin filaments 51 , which could then be analyzed in ONE microscopy. The images reproduce the known size of the actin filaments and the distance between the actin subunits, as well as providing views of the filament pitch ( Fig.…”

Section: Introductionmentioning

confidence: 99%

Visualizing proteins by expansion microscopy

Shaib

Chouaib

Chowdhury

et al. 2022

Preprint

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Fluorescence imaging is one of the most versatile and widely-used tools in biology. Although techniques to overcome the diffraction barrier were introduced more than two decades ago, and the nominal attainable resolution kept improving to reach single-digit nm, fluorescence microscopy still fails to image the morphology of single proteins or small molecular complexes, either purified or in a cellular context. Here we report a solution to this problem, in the form of one-nanometer expansion (ONE) microscopy. We combined the 10-fold axial expansion of the specimen (1000-fold by volume) with a fluorescence fluctuation analysis to achieve resolutions down to 1 nm or better. We have successfully applied ONE microscopy to image cultured cells, tissues, viral particles, molecular complexes and single proteins. At the cellular level, using immunostaining, our technology revealed detailed nanoscale arrangements of synaptic proteins, including a quasi-regular organisation of PSD95 clusters. At the single molecule level, upon main chain fluorescent labelling, we could visualise the shape of individual membrane and soluble proteins. Moreover, conformational changes undergone by the ~17 kDa protein calmodulin upon Ca2+ binding were readily observable. We could also image and classify molecular aggregates in cerebrospinal fluid samples from Parkinson's Disease (PD) patients, which represents a promising new development towards an improved PD diagnosis. ONE microscopy is compatible with conventional microscopes and can be performed with the software we provide here as a free, open-source package. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, and provides a new avenue for discoveries in biology and medicine.

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“…Sample processing for PREM was performed as described previously 96 , 97 . In brief, glutaraldehyde-fixed cells were post-fixed by sequential treatment with 0.1% tannic acid and 0.2% uranyl acetate in water, critical-point dried, coated with platinum and carbon, and transferred onto EM grids for observation.…”

Section: Methodsmentioning

confidence: 99%

Actin polymerization promotes invagination of flat clathrin-coated lattices in mammalian cells by pushing at lattice edges

et al. 2022

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Clathrin-mediated endocytosis (CME) requires energy input from actin polymerization in mechanically challenging conditions. The roles of actin in CME are poorly understood due to inadequate knowledge of actin organization at clathrin-coated structures (CCSs). Using platinum replica electron microscopy of mammalian cells, we show that Arp2/3 complex-dependent branched actin networks, which often emerge from microtubule tips, assemble along the CCS perimeter, lack interaction with the apical clathrin lattice, and have barbed ends oriented toward the CCS. This structure is hardly compatible with the widely held “apical pulling” model describing actin functions in CME. Arp2/3 complex inhibition or epsin knockout produce large flat non-dynamic CCSs, which split into invaginating subdomains upon recovery from Arp2/3 inhibition. Moreover, epsin localization to CCSs depends on Arp2/3 activity. We propose an “edge pushing” model for CME, wherein branched actin polymerization promotes severing and invagination of flat CCSs in an epsin-dependent manner by pushing at the CCS boundary, thus releasing forces opposing the intrinsic curvature of clathrin lattices.

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Imaging Cytoskeleton Components by Electron Microscopy

Cited by 20 publications

References 92 publications

Super-resolution imaging pinpoints ultrastructural changes at the node of Ranvier in patients with polyneuropathy

Super-resolution imaging pinpoints ultrastructural changes at the node of Ranvier in patients with polyneuropathy

Visualizing proteins by expansion microscopy

Actin polymerization promotes invagination of flat clathrin-coated lattices in mammalian cells by pushing at lattice edges

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